Clinical tests using antigen-antibody reactions have become increasingly important in the diagnoses of various human ailments. This type of testing, of course, also proves invaluable in blood compatibility determinations performed prior to transfusions. It not only, in general, assures the blood's suitability for the recipient (patient), but also screens for unexpected antibodies in the donor and the recipient.
Other conditions also benefit from the use of clinical tests based upon antigen-antibody reactions. These tests find important use for immunologic disease states such as immune hemolytic anemias due to drug-antidrug complexes. Other examples include autoimmune hemolytic anemia, erythroblastosis fetalis, and transfusion reactions resulting from incompatible blood.
In direct testing procedures, an antiserum containing antibodies to human immunoglobulin(s) and/or complement component(s) receives the erythrocytes undergoing testing. Agglutinated cells in the test indicate the presence immunoglobulin(s) and/or complement component(s) on the erythrocytes. The testing procedure usually includes one or more controls to insure the proper reactivity of the reagents.
In indirect testing, the patient's serum typically undergoes incubation with red blood cells. The incubation allows antibodies in the serum to attach to antigenic sites on the erythrocytes. The cells, after removal of the patient's serum by washing upon completion of incubation, undergo testing with a serum containing antibodies to immunoglobulin(s) and/or complement component(s). This step resembles the direct testing procedures discussed above. The agglutination of the erythrocytes in the last step indicates the presence of the corresponding immunoglobulin(s) and/or complement component(s) on the erythrocytes, and indicates the presence of antibodies to antigens on the erythrocyte surface. Again, the use of controls minimizes false results.
The antiserum used in the tests may take one of two forms. The classical, or polyspecific, antiserum generally contains antibodies to a wide range of immunoglobulins and complement components. In either the direct or indirect procedure, if the erythrocytes have become sensitized (coated) with any of these immunoglobulins or complement components, agglutination will occur and give a positive test result. The specific immunoglobulin(s) and/or complement component(s) that produced the positive result, however, remain unknown with the use of this polyspecific type of antiserum.
Most, if not all, of the tests employing polyspecific antiserum will provide additional pertinent information if repeated with monospecific antisera. In comparison to the polyspecific antiserum, these latter reagents contain an antibody for a single immunoglobulin class or complement component. Agglutination occurs, of course, only if the erythocytes' surfaces contain the specific immunoglobulin or complement component corresponding to the antibody in the antiserum.
The reliability of the direct and indirect testing procedures depends heavily upon the concentrations of the antibodies in both the polyspecific and monospecific antisera. Moreover, the purity (degree of monospecificity) of the single antibody in each of the monospecific antisera represents an important criterion in assuring the accuracy of the results of analyses employing them.
However, the antibodies in the antisera represent esoteric proteins which may readily undergo denaturation and degeneration. This can occur as a result merely of the passage of time or of even slightly improper storage conditions. These deleterious occurrences can unfavorably alter the nature of either the polyspecific or monospecific antisera.
Thus, the strengths of the antibodies in the antisera must remain known in order for the results of tests employing the antisera to have validity. This required knowledge involves two separate aspects for any particular antiserum. The specific immunoglobulin(s) and/or complement component(s) with which its antibodies will react represents a first crucial aspect of the antiserum. Furthermore, the reactive strength of the antiserum towards its corresponding protein must be known in order to provide meaningful test results. And, the reactive strengths of a polyspecific antiserum to each of its corresponding immunoglobulins and complement components must remain controlled to avoid inaccurate results.
Determining the antibodies and their reactive strengths in the antiserum does not represent a classical chemical problem. An antibody molecule generally can react with only a specific single amino-acid group on an immunoglobulin molecule or complement molecule. Thus, different antibodies react with different amino-acid combinations and, therefore, different immunoglobulins or complement components. However, antibodies with different specificities have basic chemical structures which are very similar. Present chemical knowledge lacks the tools to differentiate between the different antibodies for the different immunoglobulins and complement components on the basis of molecular structure. Yet, the antibodies that correspond to the different proteins do, in fact, possess differencs. They display reactivity only with their single corresponding immunoglobulin or complement component.
Thus, the only readily definable difference between the different antibodies centers upon the reactivity of each with their separate protein; the only indication of the presence of an antibody for a particular immunoglobulin, for example, requires the reaction of that antibody with that immunoglobulin. One commonly used method for detecting this reaction involves the agglutination of erythrocytes to which the particular protein or immunoglobulin has attached itself. For example, by this method determining the presence of the antibody for the IgG immunoglobulin in an antiserum requires reacting that antiserum with erythrocytes having IgG molecules attached to them. Any resulting agglutination and the strength of that agglutination will show the presence and reactive strength of the antibody in the antiserum.
Thus, the need for erythrocytes having a single attached immunoglobulin class or complement component becomes clear. Such cell preparations would assume an absolutely crucial role in determining the presence and reactive strengths of antibodies in both polyspecific and monospecific antisera. These cell preparations would thus assure the suitability of these antisera for their important clinical tests.
While the need for red cell preparations having a single immunoglobulin class or complement component has become clear, their preparation has eluded success. Various attempts at making red blood cell suspensions having a single attached protein have not achieved their objective. For example, the "low ionic-strength method" of J. A. Moore and H. Chaplin, Jr., in their article "Anti-C3d Antiglobulin Reagents. II. Preparation of an Antiglobulin Serum Monospecific for C3d" appearing in Transfusion, 14:416 (1974), attempts to produce erythrocytes having only the C3 complement component attached. However, as shown in the article of G. Garratty and L. Petz, "The Significance of Red Cell Bound Complement Components in Development of Standards and Quality Assurance for the Anti-Complement Components of Antiglobulin Sera" appearing in Transfusion, 16:297 (1976), these procedures result in cells sensitized with detectable immunoglobulins as well as other complement components. Moreover, the reactive strength of the desired complement component is not always at the desired level. Thus, the need for cell suspensions having a single attached protein has remained unfilled.